Linde Dolores, Macias Isabel, Fernández-Arrojo Lucía, Plou Francisco J, Jiménez Antonio, Fernández-Lobato María
Centro de Biología Molecular Severo Ochoa (CSIC/UAM), Departamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Appl Environ Microbiol. 2009 Feb;75(4):1065-73. doi: 10.1128/AEM.02061-08. Epub 2008 Dec 16.
An extracellular beta-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70 degrees C) and thermostability (with a T(50) in the range 66 to 71 degrees C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-beta-(2-->1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the k(cat)/K(m) ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial beta-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter(-1). In addition, we isolated and sequenced the X. dendrorhous beta-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant beta-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.
对来自酵母类胡萝卜素红酵母（Xanthophyllomyces dendrorhous）的一种胞外β-呋喃果糖苷酶进行了生化、分子和系统发育特征分析。这种酶是一种糖蛋白，估计分子量为160 kDa，其中N-连接的碳水化合物占总质量的60%。它在pH 5.0至6.5时表现出最佳活性，其嗜热性（在65至70摄氏度时活性最高）和热稳定性（T(50)在66至71摄氏度范围内）高于大多数酵母转化酶。该酶能够水解果糖基-β-(2→1)-连接的碳水化合物，如蔗糖、1-蔗果三糖或蔗果四糖，尽管其由k(cat)/K(m)比值定义的催化效率表明它水解蔗糖的效率比1-蔗果三糖高约4.2倍。与其他微生物β-呋喃果糖苷酶不同，来自类胡萝卜素红酵母的这种酶产生新蔗果四糖作为主要的转糖基化产物——一种潜在的新型双歧因子三糖。使用41%（重量/体积）的蔗糖溶液，达到的最大低聚果糖浓度为65.9 g·L⁻¹。此外，我们分离并测序了类胡萝卜素红酵母β-呋喃果糖苷酶基因（Xd-INV），表明它编码一个推定的由595个氨基酸组成的成熟多肽，并且与其他真菌、酵母和植物β-呋喃果糖苷酶具有显著的同源性，这些酶都是糖基水解酶家族32的成员。我们证明Xd-INV可以在功能上互补酿酒酵母（Saccharomyces cerevisiae）的suc2突变，最后，还获得了基于拟南芥同源转化酶的新酶的结构模型。
